Medical diagnostic imaging has evolved as an important non-invasive tool for the evaluation of pathological and physiological processes. Presently, nuclear magnetic resonance imaging (“MRI”) and computerized tomography (“CT”) are two of the most widely used imaging modalities. Although both MRI and CT can be performed without the administration of contrast agents, the ability of many contrast enhancement agents to enhance the visualization of internal tissues and organs has resulted in their widespread use.
Principles of Magnetic Resonance Imaging and Contrast Enhancement Agents
Proton MRI is based on the principle that the concentration and relaxation characteristics of protons in tissues and organs can influence the intensity of a magnetic resonance image. Contrast enhancement agents that are useful for proton MRI effect a change in the relaxation characteristics of protons which can result in image enhancement and improved soft-tissue differentiation. Different classes of proton MR imaging agents include paramagnetic metal chelates and nitroxyl spin labeled compounds.
MRI is a diagnostic and research procedure that uses a large, high-strength magnet and radio-frequency signals to produce images. The most abundant molecular species in biological tissues is water. It is the quantum mechanical “spin” of the water proton nuclei that ultimately gives rise to the signal in all imaging experiments. In an MRI experiment, the sample to be imaged is placed in a strong static magnetic field (on the order of 1–12 Tesla) and the spins are excited with a pulse of radio frequency (“RF”) radiation to produce a net magnetization in the sample. Various magnetic field gradients and other RF pulses then act on the spins to code spatial information into the recorded signals. The basic MRI experiment can be described, in one frame of reference, as follows. Pre-RF pulse spins can be thought of as collectively aligned along the Z-axis of a Cartesian coordinate system; application of one or a sequence of RF pulses “tip” the spins into the X-Y plane, from which position they will spontaneously relax back to the Z-axis. The relaxation of the spins is recorded as a function of time. Using this basic experiment, MRI is able to generate structural information in three dimensions in a relatively short period of time.
MR images are typically displayed on a gray scale with the color black representing the lowest measured intensity and white representing the highest measured intensity (I). This measured intensity is obtained by applying the formula I=C*M, where C is the concentration of spins (in an MRI experiment, this represents the water concentration) and M is a measure of the magnetization in the sample present at time of the measurement. Although variations in water concentration (C) can give rise to contrast in MR images, it is the strong dependence of the rate of change in the magnetization (M) on local environment that is the major source of variation in image intensity in an MRI experiment.
Two characteristic relaxation times are implicated in magnetic relaxation, the basis for MRI. T1 is defined as the longitudinal relaxation time, and is also known as the spin lattice relaxation time (1/T1 is a rate constant, R1, the spin-lattice relaxation rate constant). T2 is known as the transverse relaxation time, or spin-spin relaxation mechanism, which is one of several contributions to T2 (1/T2 is also a rate constant, R2, the spin-spin relaxation rate constant). T1 and T2 have inverse and reciprocal effects on image intensity, with image intensity increasing either by shortening the T1 or lengthening the T2.
In order to increase the signal-to-noise ratio (“SNR”), a typical MR imaging scan (RF and gradient pulse sequence and data acquisition) is repeated at a constant rate for a predetermined number of times and the data is subsequently averaged. The signal amplitude recorded for any given scan is proportional to the number of spins that have decayed back to equilibrium in the time period between successive scans. Thus, regions with rapidly relaxing spins (i.e. those regions comprising spins having short T1 values) will recover all of their signal amplitude between successive scans. The measured intensities of the regions with long T2 and short T1 will reflect the spin density, which correlates with the region's water content. Regions with long T1 values, as compared to the time between scans, will progressively lose signal (i.e. the signal linewidth will broaden and “flatten out”) until a steady state condition is reached. At the steady state condition, these regions will appear as darker regions in the final image. In extreme situations, the linewidth can be so large that the signal is indistinguishable from background noise.
Clinical MR imaging takes advantage of the fact that water relaxation characteristics vary from tissue to tissue, and this tissue-dependent relaxation effect provides image contrast, which in turn allows the identification of various distinct tissue types. Additionally, the MRI experiment can be set up so that regions of a sample with short T1 values and/or long T2 values are preferentially enhanced. Experiments so designed are known as T1-weighted and T2-weighted imaging protocols.
There is a rapidly growing body of literature demonstrating the clinical effectiveness of paramagnetic contrast agents. Paramagnetic contrast agents serve to modulate T1 and/or T2 values, and are typically designed with regard to a given metal nucleus, which is usually selected based on its effect on relaxation. The capacity to differentiate between regions or tissues that can be magnetically similar but histologically different is a major impetus for the preparation of these agents. Paramagnetic contrast agents provide additional image contrast, and thus enhanced images, of those areas where the contrast agent is localized. For example, contrast agents can be injected into the circulatory system and used to visualize vascular structures and abnormalities (See, e.g., U.S. Pat. No. 5,925,987), or even intracranially to visualize structures of the brain.
When designing contrast agents for use in MRI experiments, strict attention must be given to a variety of properties that will ultimately affect the physiological applicability of the agent, as well as the ability of the agent to provide contrast enhancement in an MRI image. Two fundamental properties that must be considered are a) biocompatibility, and b) proton relaxation enhancement. Biocompatability is influenced by several factors including toxicity, stability (thermodynamic and kinetic), pharmacokinetics and biodistribution. Proton relaxation enhancement and relaxivity is chiefly governed by the choice of metal employed in the agent, the rotational correlation times and the accessibility of the metal to surrounding water molecules, which permits the rapid exchange of metal-associated water molecules with the bulk solvent.
The measured relaxivity of the contrast agent is dominated by the selection of the metal atom. Paramagnetic metal ions, as a result of their unpaired electrons, act as potent relaxation enhancement agents. They decrease the T1 relaxation times of nearby spins, exhibiting an r6 dependency, where r is the distance between the two nuclei. Some paramagnetic ions decrease the T1 without causing substantial linebroadening, for example gadolinium(III) (“Gd(III)”), while others induce drastic linebroadening, for example, superparamagnetic iron oxide. The mechanism of T1 relaxation is generally a through-space dipole-dipole interaction between the unpaired electrons of a metal atom with an unpaired electron (the paramagnet) and those water molecules not coordinated to the metal atom that are in fast exchange with water molecules in the metal's inner coordination sphere.
The shortening of proton relaxation times by Gd(III) is mediated by dipole-dipole interactions between its unpaired electrons and adjacent water protons. The effectiveness of Gd(III)'s magnetic dipole drops off very rapidly as a function of its distance from these protons. Consequently, the protons which are relaxed most efficiently are those which are able to enter Gd(III)'s first or second coordination spheres during the interval between the RF pulse and signal detection. By way of example, regions associated with a Gd(III) ion having proximate water molecules appear bright in an MR image, while the normal aqueous solution appears as dark background when the time between successive scans in the experiment is short, for example in a T1 weighted image.
Conversely, localized T2 shortening caused by superparamagnetic particles is believed to be due to the local magnetic field inhomogeneities associated with the large magnetic moments of these particles. Regions associated with a superparamagnetic iron oxide particle appear dark in an MR image where the normal aqueous solution appears as high intensity background if the echo time (“TE”) in a spin-echo pulse sequence experiment is long, for example in a T2-weighted image.
The lanthanide atom Gd(III) has generally been chosen as the metal atom for contrast agents because it has a high magnetic moment (μ2=63 BM2), a symmetric electronic ground state, S8, and the largest paramagnetic dipole and the greatest paramagnetic relaxivity of any element. Gd(III) can be chelated with any of a number of substances to render the Gd(III) complex nontoxic, such as diethylenetriamine-pentaacetic acid (“DTPA”), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (“DOTA”), and derivatives thereof. See, U.S. Pat. Nos. 5,155,215; 5,087,440; 5,219,553; 5,188,816; 4,885,363; 5,358,704; 5,262,532; and Meyer et al., (1990) Invest. Radiol. 25:S53.
The stability constant (K) for the Gd-DTPA complex is very high (logK=22.4) and is more commonly known as the formation constant, expressed by the qualitative observation that the higher the value of logK, the more stable the complex. This thermodynamic parameter indicates that the fraction of Gd(III) ions that are in the unbound state will be quite small, and should not be confused with the rate (kinetic stability) at which the loss of metal occurs.
The water soluble chelate Gd-DTPA is stable, nontoxic, and one of the most widely used contrast enhancement agents in experimental and clinical imaging research. It was approved for clinical use in adult patients in June of 1988. Gd-DTPA is an extracellular agent that accumulates in tissue by perfusion-dominated processes. Image enhancements achieved using Gd-DTPA are well documented for a variety of applications including visualizing blood-brain barrier disruptions caused by space-occupying lesions and detection of abnormal vascularity. (Runge et al., (1991) Magn, Reson. Imaging 3:85; Russell et al., (1989) Am. J. Roentgenol. 152:813; and Meyer et al., (1990) Invest Radiol 25:S53). It has also been applied to the functional mapping of the human visual cortex by defining regional cerebral hemodynamics (Belliveau et al., (1991) Science 254: 719).
Another chelator used in Gd-based contrast agents is the macrocyclic ligand DOTA. The Gd-DOTA complex has been thoroughly studied in laboratory tests involving animals and humans. The complex is conformationally rigid, has an extremely high formation constant (logK=28.5) and, at physiological pH, possesses very slow dissociation kinetics. The Gd-DOTA complex was approved as an MRI contrast agent for use in adults and infants in France and has been administered to over 4,500 patients.
Tissue Transglutaminase
The extracellular matrix (“ECM”) plays an important role in the regulation of tumor growth, metastasis and angiogenesis. To generate a new EC matrix, tumors are known to elicit wound-healing responses from the host tissues resulting in formation of granulation tissue at the advancing margins of the tumor. Tissue transglutaminase (“TG”) is one of a family of crosslinking enzymes expressed in different forms in a variety of biological systems. TG is a calcium dependent enzyme that covalently crosslinks a wide variety of ECM proteins, producing a protease resistant matrix. TG is reported to be expressed at sites of wound healing and inflammation (Haroon et al., (1999a) FASEB J. 13:1787–95; Haroon et al., (1999b) Lab. Invest. 79:1679–86; Hettasch et al., (1996) Lab. Invest. 75:637–45) and, therefore, appears to play a role in the host response to these conditions. TG is also known to be expressed and active at sites of tumor progression.
TG catalyzes the formation of ε-(γ-glutamyl) lysine bonds (isopeptide bonds) between peptide bound glutamine residues and the primary amine group of various amines (Folk, (1983) Adv. Enzymol. RAMB 54: 1–56). These isopeptide bonds are stable and more resistant to proteolytic degradation than non-covalent linkages. The covalent crosslinking reaction increases the resistance of proteins to chemical, enzymatic and physical disruption (Greenberg et al., (1991) FASEB J. 5:3071–77). The list of proteins that are crosslinked by TG is extensive and includes extracellular adhesive proteins such as fibronectin, collagen, fibrinogen, fibrin, laminin/nidogen, osteoporin and vitronectin to name a few. See, Aeschlimann & Paulsson, (1991) J. Biol. Chem. 266:15308–17; Greenberg et al., (1987) Blood 70:702–9; Hohenadl et al., (1995) J. Biol. Chem. 270:23415–20; Sane et al., (1988) Biochem. Biophys. Res. Commun. 157:115–20. In addition to influencing matrix stability by producing stable crosslinks between matrix proteins, TG has been implicated in numerous other interactions to stabilize the ECM. TG has been shown to regulate the conversion of latent TGF β, a cytokine that can modify epithelial growth, enhance synthesis of various ECM proteins and inhibitors of metalloproteases (Clark & Coker, (1998) Int. J. Biochem. Cell Biol. 30: 293–98; Kojima et al., (1993) J. Cell Biol. 121: 439–48; Pepper, (1997) Cytokine Growth Factor Rev. 8: 21–43), to its active form. TG itself is induced by injury cytokines such as TNFα, TGFβ and IL-6 (George et al., (1990) J. Biol. Chem. 265:11098–104; Ikura et al., (1994) Biosci. Biotechnol. Biochem. 58: 1540–41; Kuncio et al., (1998) Am. J. Physiol. 274: G240–45). The TG can also crosslink elafin and PAI-2, potent inhibitors of elastase and plasmin respectively, to ECM thus providing anti-protease capability to the matrix (Jensen et al., (1993) Eur. J. Biochem. 214:141–46; Nara et al., (1994) J. Biochem. (Tokyo) 115: 441–48). Fibroblasts transfected with TG have been shown to have a distinct spread morphology and increased resistance to protease digestion (Gentile et al., (1992) J. Cell Biol. 119: 463–74).
Plasma Factor XIII and fXIIIa 
Plasma factor XIII (also known as fibrin stabilizing factor, fibrinoligase, or plasma transglutaminase) is a plasma glycoprotein that circulates in blood as a zymogen (Mr≅320 kD) complexed with fibrinogen (Greenberg & Shuman, (1982) J. Biol. Chem. 257: 6096–6101). The plasma factor XIII zymogen is a tetramer consisting of two a subunits (Mr≅75 kD) and two b subunits (Mr≅80 kD) (Chung et al., (1974) J. Biol Chem. 249: 940–50), having an overall structure designated as a2b2. The a subunit contains the catalytic site of the enzyme, while the b subunit is thought to stabilize the a subunit or to regulate the activation of factor XIII (Folk & Finlayson, (1977) Adv. Prot. Chem. 31: 1–133; Lorand et al., (1974) Biochem. Biophys. Res. Comm. 56: 914–922). The amino acid sequences of the a and b subunits are known (Ichinose et al., (1986a) Biochem. 25: 4633–38; Ichinose et al., (1986b) Biochem. 25: 6900–906). Plasma factor XIII occurs in placenta and platelets as an a2 homodimer.
In vivo, activated factor XIII (“fXIIIa”) catalyzes crosslinking reactions between other protein molecules. Factor XIIIa, a sister enzyme of tissue TG discussed above, catalyzes a number of covalent crosslinking reactions of fibrin in blood clots. These covalent fibrin crosslinking reactions render blood clots mechanically stable and greatly increase clot resistance to plasma degradation (fibrinolysis).
During the final stages of blood coagulation, thrombin converts the plasma factor XIII zymogen to an intermediate form (a′2 b2), which then dissociates in the presence of calcium ions to produce factor XIIIa, a homodimer of a′ subunits. Placental factor XIII is also activated upon cleavage by thrombin. Factor XIIIa (“fXIIIa”) is a transglutaminase that catalyzes the crosslinking of fibrin polymers through the formation of intermolecular ξ(δ-glutamyl) lysine bonds, thereby increasing clot strength (Chen & Doolittle, (1970) Proc. Natl. Acad. Sci. U.S.A. 66: 472–79; Pisano et al., (1972) Ann. N.Y. Acad. Sci. 202: 98–113). This crosslinking reaction requires the presence of calcium ions (Lorand et al., (1980) Prog. Hemost. Throm. 5: 245–290; Folk & Finlayson, (1977) Adv. Prot. Chem. 31: 1–133). Factor XIIIIa also catalyzes the crosslinking of the δ-chain of fibrin to α2-plasmin inhibitor and fibronectin, as well as the crosslinking of collagen and fibronectin, which might be related to wound healing (Sakata & Aoki, (1980) J. Clin. Invest 65: 290–97; Mosher, (1975) J. Biol. Chem. 250: 6614–21; Mosher & Chad, (1979) J. Clin. Invest 64: 781–787; Folk & Finlayson, (1977) Adv. Prot. Chem. 31: 1–133; Lorand et al., (1980) Prog. Hemost. Throm. 5: 245–90). The covalent incorporation of α2-plasmin inhibitor into the fibrin network might increase the resistance of the clot to lysis (Lorand et al., (1980) Prog. Hemost. Throm. 5: 245–90).
Simultaneous with catalyzing γ chain crosslinking, fXIIIa catalyzes crosslinking of fibrin alpha (“α”) chains to a plasminogen inhibitor, α2-PI. This α2-PI incorporation into a crosslinked clot gives a clot immediate protection against fibrinolysis. Subsequently, and at much slower rates (up to 6 days) in a clot, fXIIIa catalyzes lateral crosslinking of a chains to form clusters of five to seven γ chains. In addition, fXIIIa catalyzes (i) a chain crosslinking to γ dimers; and (ii) γ dimer crosslinking with other γ chains to form γ trimers and tetramers. In general, the older the clot is, the greater amount of the fibrin crosslinking is present and, consequently, the greater resistance the clot is to lysis.
Blood Clot Imaging
The ability to detect the presence of clots, and the ability to monitor their formation and dissolution, can eliminate a potentially life-threatening condition, particularly in a patient recovering from a myocardial infarction (heart attack). After a clot that has caused a myocardial infarction has been lysed, a second or third clot can reformat the same site, which can become more life-threatening than the formation of the first clot. This is because, when the first occluding clot is lysed, it is common that the clot does not entirely dissolve. As blood flow is restored over the undissolved portion of the clot, some blood constituents cause platelet deposition and promote further thrombosis. When this happens, the natural hemostatic balance (the balance between clot formation and clot degradation) is shifted from fibrinolysis toward thrombosis. The surface of any undissolved portion of the first clot is intrinsically thrombogenic, and more fibrin is often deposited before dissolution of the first clot is complete. This newly deposited fibrin gives the older, undissolved portion of the clot an enhanced opportunity to become more crosslinked. As a clot persists over time, more fibrin crosslinking occurs, and the clot becomes denser and more resistant to fibrinolysis, until a very mature, fibrin-dense clot is formed.
A patient suffering from a heart attack can be treated with agents to assist in restoring coronary blood flow. However, the patient still runs the risk of clot reformation and a subsequent occlusion of blood vessels. It is therefore desirable to monitor blood clot formation and dissolution in vivo. The ability to monitor the status of blood clots reduces the chance that a patient can suffer from the occurrence or re-occurrence of occluded blood vessels. A number of patents disclose the in vitro detection of blood clots (See. e.g., U.S. Pat. Nos. 6,022,747; 4,797,369) and the in vitro measurement of blood clot mass (See, e.g., U.S. Pat. No. 5,441,892). The in vivo detection of blood clots, however, has been elusive. In vivo detection of blood clots would be useful as a preventative measure, and would provide an in vivo a therapeutic and research tool for determining the effect of developed and candidate inhibitors of clot formation and effectors of clot dissolution.
Imaging Wound Healing
The ability to monitor and image internal wound healing is vital, yet has been elusive. In medical procedures wherein the wound healing process is not immediately observable, such as in a bowel resection or other surgical procedure, it is critical for the health care practitioner to be able to monitor the healing process. If such a wound is not healing properly, a potentially life-threatening condition can develop in a subject.
When a surgical procedure is performed on a structure that can be visually observed, an incision can easily be monitored for proper healing via visual inspection and other known methods, and there is no immediate need for forming an image of the wound healing structure. When an internal operation is performed, however, it is much more difficult to assess wound healing. Internal wounds, obviously, cannot be visually inspected. Techniques currently available to health care professionals for monitoring imagine internal wound healing are both limited and imprecise, or entirely unavailable. Additionally, known techniques cannot provide a detailed image of an internal wound healing structure.
Imaging Tumor Boundaries and Monitoring Tumor Growth and Remission
It is also important for a heath care practitioner to be able to identify the discrete boundaries of a tumor. Tumor growth and remission can be monitored by determining the size of the tumor, prior and subsequent to a treatment; the boundaries of the tumor can be used as an indicator of size. In a clinical setting, an observed reduction in tumor size can be indicative of the success of a particular treatment.
Associated with tumor growth, and a contributing factor to tumor size, is the phenomenon of angiogenesis. Under normal physiological conditions, humans or animals undergo angiogenesis only in very specific restricted situations. For example, angiogenesis is normally observed in wound healing, fetal and embryonal development and formation of the corpus luteum, endometrium and placenta. Uncontrolled angiogenesis is associated with tumor metastasis. Folkman, (1995) New Engl. J. Med. 28:333(26), 1757–63. Indeed, tumors have been loosely characterized in the art as wounds that do not heal. Dvorak et al., (1987) Lab. Invest. 57(6): 673–86.
Imaging Angiogenic Tissue
Both controlled and uncontrolled angiogenesis are thought to proceed in a similar manner. Endothelial cells and pericytes, surrounded by a basement membrane, form capillary blood vessels. Angiogenesis begins with the erosion of the basement membrane by enzymes released by endothelial cells and leukocytes. The endothelial cells, which line the lumen of blood vessels, then protrude through the basement membrane. Angiogenic stimulants induce the endothelial cells to migrate through the eroded basement membrane. The migrating cells form a “sprout” off the parent blood vessel, where the endothelial cells undergo mitosis and proliferate. The endothelial sprouts merge with each other to form capillary loops, creating the new blood vessel.
Persistent, unregulated angiogenesis occurs in a multiplicity of disease states, and abnormal growth by endothelial cells supports the pathological damage seen in these conditions. The diverse pathological disease states in which unregulated angiogenesis is present have been grouped together as angiogenic-dependent or angiogenic-associated diseases.
It is also recognized that angiogenesis plays a major role in the metastasis of a cancer. If this angiogenic activity could be repressed or eliminated, then the tumor, although present, would not grow. In lieu of modulating angiogenesis, it is desirable to observe tumor growth and size by monitoring angiogenesis, and conversely, to observe angiogenesis in a tumor by monitoring tumor growth and size. It can also be valuable to monitor angiogenesis in non-tumor tissues, and ideally, a system designed to monitor and/or image angiogenic tumor tissue will be equally applicable to monitor and/or image angiogenic non-tumor tissue.
Detection and monitoring of tumor growth and remission is vital for the effective diagnosis and treatment of cancer. Current methods for detecting tumor growth and regression using CT scan, positron emission tomography (“PET”), optical imaging and MRI are limited in their ability to distinguish between normal and tumor tissue. Additionally, the ability to image blood clot and their formation finds application in a variety of different scenarios. Blood clot imaging is presently limited by a variety of obstacles, one of which is the inability to selectively image blood clots over other tissues and structures. Further, there is no currently available method of monitoring internal wound healing, nor is there an adequate method of monitoring angiogenic activity in tumor and non-tumor tissue. What is needed, therefore, is a non-invasive method of monitoring tumor growth or regression, blood clot formation and dissolution, angiogenesis and wound healing in a subject that offers superior sensitivity, relative to the currently available methods.